JP4387445B2 - Mucosal agent and method for producing the same - Google Patents

Description

  The present invention relates to an agent for applying to mucosa containing a hydrophobic group-linked glycosaminoglycan as an active ingredient, and a method for producing the same.

BACKGROUND ART Conventionally, hyaluronic acid, which is a typical glycosaminoglycan (hereinafter referred to as “GAG”), is known as a substance having a healing effect on mucosal disorders such as inflammation and damage. (For example, Patent Document 1). However, in the cornea, oral and nasal mucosa, and conjunctiva in contact with the outside world; and in the mucous membrane of the bladder, the mucosal surface is washed with secretions and excretion such as tears, saliva and urine, and foreign objects are removed. . Therefore, there has been a demand for a derivative of GAG that retains the original drug efficacy of GAG and that exhibits high retention in these mucosal tissues.

  On the other hand, photoreactive hyaluronic acid is known in which water solubility is increased by bonding a photocrosslinking group such as cinnamic acid to hyaluronic acid and further adding an alkali treatment thereto (for example, Patent Document 2). ). This photoreactive hyaluronic acid is provided by bonding a photocrosslinking group such as cinnamic acid to hyaluronic acid in order to provide a medical material such as an anti-adhesion material by providing photocrosslinking. It is not intended to enhance retention in the tissue.

[Patent Document 1] JP-A-1-238530 [Patent Document 2] JP-A-2002-249501

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention An object of the present invention is to provide a mucosal application agent that exhibits excellent retention and pharmacological effects in the mucosa.

Means for Solving the Problems As a result of extensive research to solve the above problems, the present inventors have obtained “hydrophobic group-binding GAG” obtained by binding a hydrophobic group to GAG via a binding chain. However, this GAG retains the healing effect inherent to GAG against mucosal disorders such as inflammation and injury and is applied to it. In this case, the present invention was completed.

  The present invention relates to a mucosal application agent containing glycosaminoglycan (GAG) into which a hydrophobic group has been introduced via a binding chain.

EFFECT OF THE INVENTION Since the agent for applying to mucosa of the present invention can stay in the affected area for a long time by exhibiting high retention in the mucosa, it can be continuously cured against mucosal disorders such as inflammation and damage even by low frequency administration. The effect can be demonstrated.

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in more detail below by the best mode for carrying out the invention.
As used herein, an alkyl group refers to a linear or branched aliphatic hydrocarbon group having the number of carbon atoms described. An alkenyl group refers to a straight or branched aliphatic hydrocarbon group having the stated number of carbon atoms having at least one double bond. An alkynyl group refers to a straight or branched aliphatic hydrocarbon group having the number of carbon atoms described having at least one triple bond.

  An aryl group refers to a monocyclic or polycyclic aromatic hydrocarbon group having 6 to 20 carbon atoms as ring-constituting atoms. The heteroaryl group is a monocyclic or polycyclic aromatic hydrocarbon having 3 to 20 carbon atoms and one or more heteroatoms selected from nitrogen, sulfur and oxygen atoms as ring-constituting atoms. Refers to the group.

  An arylalkyl group refers to an alkyl group as defined above substituted with an aryl group as defined above. An arylalkenyl group refers to an alkenyl group as defined above substituted with an aryl group as defined above. An arylalkynyl group refers to an alkynyl group as defined above substituted with an aryl group as defined above.

  The amino acid group refers to a group derived from a natural or synthetic amino acid by losing a carboxyl group, an amino group or a hydroxyl group by a chemical bond.

  As used herein, the term “treatment” includes prevention of mucosal damage, inhibition of progression (prevention of deterioration), improvement (reduction) and treatment. “Mucosal disorder” means a state in which the form, characteristics and function that the mucous membrane should have are impaired in some way. For example, conditions such as damage, defect, erosion, inflammation, ulcer and dryness can be mentioned as mucosal disorders.

  The GAG having a hydrophobic group introduced via a linking chain, which is contained as an active ingredient in the agent for applying to mucosa of the present invention, is derived from a hydrophobic compound having a property that is hardly soluble in water and soluble in oil. Any GAG may be used as long as it is bound to a hydrophobic group. This hydrophobic group is bonded to GAG via a bonding chain. As will be described later, it is not necessary that all structural units of GAG have a hydrophobic group bonded thereto.

1) GAG
GAG in GAG having a hydrophobic group introduced via a linking chain, which is included in the agent for applying to mucosa of the present invention, is an acidic polysaccharide having a repeating long chain structure of a disaccharide consisting of an amino sugar and uronic acid (or galactose). . Examples of such GAGs include hyaluronic acid, chondroitin, chondroitin sulfate, heparin, heparan sulfate, dermatan sulfate, and keratan sulfate. Among these, hyaluronic acid is preferable. These GAGs may be pharmaceutically acceptable salts thereof. Examples of such salts include sodium salts, potassium salts, magnesium salts and calcium salts, among which sodium salts are preferred. Therefore, GAG in the agent for applying to mucosa of the present invention is most preferably sodium hyaluronate. The origin of GAG is not particularly limited, and GAG may be derived from animals or microorganisms, or may be chemically synthesized. For example, when using sodium hyaluronate, the thing derived from a chicken crown can be illustrated. The molecular weight of GAG is not particularly limited, but its weight average molecular weight is preferably 200,000 to 3,000,000, more preferably 500,000 to 2,000,000, and most preferably 600,000 to 1, 200,000. When using hyaluronic acid or a pharmaceutically acceptable salt thereof, its weight average molecular weight is preferably 200,000 to 3,000,000, more preferably 500,000 to 2,000,000, and most Preferably it is 600,000-1,200,000.

2) Hydrophobic group The hydrophobic group in GAG into which the hydrophobic group is introduced via the binding chain, which is included in the agent for applying to mucosa of the present invention, is a compound in which the hydrophobic group is hardly soluble in water and soluble in oil. As long as it is derived from, it is an arbitrary group. Examples of such groups are alkyl groups having 2 to 18 carbon atoms, alkenyl groups having 2 to 18 carbon atoms, alkynyl groups having 2 to 18 carbon atoms, aryl groups, heteroaryls Groups, arylalkyl groups, arylalkenyl groups, arylalkynyl groups and amino acid groups.

  Examples of the alkyl group having 2 to 18 carbon atoms include methyl, ethyl, n-propyl, i-propyl, n-butyl, sec-butyl, t-butyl, n-pentyl, t-pentyl, isopentyl, neopentyl, Mention may be made of n-heptyl, 5-methylhexyl, 4,4-dimethyl-pentyl, 1,1-dimethyl-pentyl and n-octyl. Among these, an alkyl group such as n-butyl having 2 to 6 carbon atoms can be preferably exemplified.

  Examples of the alkenyl group having 2 to 18 carbon atoms include vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-methyl-1-propenyl, 1-methyl-1-propenyl, 1-pentenyl, 3- Mention may be made of methyl-2-butenyl, 1-hepten-1-yl and 2-hepten-1-yl. Among these, an alkenyl group having 2 to 6 carbon atoms such as 1-butenyl can be preferably exemplified.

  Alkynyl groups having 2 to 18 carbon atoms include ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-heptynyl, 2-heptynyl and 3-heptynyl. Can be mentioned. Among these, an alkynyl group having 2 to 6 carbon atoms, such as 1-butynyl, can be preferably exemplified.

Aryl groups can include groups such as phenyl, naphthyl, anthryl and phenanthryl.
Heteroaryl groups can include groups such as furyl, thionyl, thiazolyl, oxazolyl, pyrrolyl, pyridyl, pyrimidinyl and indolyl.
Arylalkyl groups include groups such as benzyl, phenethyl, naphthylmethyl and naphthylethyl.
Arylalkenyl groups can include groups such as 2-phenyl-ethenyl and p-aminophenylethenyl.
Arylalkynyl groups can include groups such as 2-phenyl-ethynyl and p-aminophenylethynyl.

  Amino acid groups include aliphatic amino acids such as glycine, alanine and β-alanine; branched chain aliphatic amino acids such as leucine, isoleucine and valine; aromatic amino acids such as phenylalanine and tyrosine; and tryptophan and histidine. Mention may be made of groups derived from heterocyclic amino acids.

  These hydrophobic groups may be mono- or polysubstituted with groups such as hydroxyl, carboxyl, cyano, amino (which may be mono- or disubstituted with the above alkyl), nitro, oxo and alkylcarbonyloxy. Good.

Among the hydrophobic groups, an aryl group, an arylalkyl group, an arylalkenyl group, and an arylalkynyl group, which are hydrophobic groups including an aryl group, can be preferably mentioned, and substituted with an arylalkenyl group and an alkylcarbonyloxy group Particularly preferred are the aryl groups formed. Specifically, phenylethenyl and p-aminophenylethenyl can be used as such arylalkenyl groups. As the aryl group, a group such as CH ₃ — (CH ₂ ) ₁ —COO-phenyl (wherein l represents 0 or an integer of 1 to 18) can be preferably used.

  These hydrophobic groups also have functions such as UV absorption capability by having a double bond in the hydrophobic group, as shown by the functional groups such as phenyl-ethenyl exemplified above in the hydrophobic group. You may have. For example, when the agent for applying to mucosa of the present invention is used as an eye drop described later, an eye drop having a function of effectively absorbing harmful ultraviolet rays is obtained by using a group having ultraviolet absorbing ability as a hydrophobic group. Can do. Further, for example, the agent for applying to mucosa of the present invention is used to treat corneal epithelial layer disorders such as corneal epithelial disease (dry eye), keratoconjunctivitis, punctate superficial keratitis (SPK), corneal epithelial erosion, corneal epithelial defect and corneal tumor. When used for treatment, by using a group having an ability to absorb ultraviolet rays as a hydrophobic group, it is possible to provide a mucosal application agent that has a pharmacological action on the above-mentioned disorder together with a function of effectively absorbing harmful ultraviolet rays. it can. As the group having ultraviolet absorbing ability, for example, an arylalkenyl group having a conjugated double bond exemplified by the above-mentioned 2-phenyl-ethenyl and p-aminophenylethenyl is preferable. When the agent for applying mucosa of the present invention is used for corneal injury, “GAG having a hydrophobic group introduced” as an active ingredient of the agent for applying mucosa of the present invention is described later when it is made into an aqueous solution of 0.1% by weight. When measuring the ultraviolet transmittance by the method described in the Examples, it is preferable to block the transmission of ultraviolet rays having a wavelength of 200 to 300 nm by 70 to 100%. Such hydrophobic groups having UV absorbing ability can preferably include arylalkenyl groups such as 2-phenyl-ethenyl and p-aminophenylethenyl.

3) Bonded chain In the GAG into which a hydrophobic group is introduced via a bond chain, which is included in the agent for applying to mucosa of the present invention, the GAG is bonded to the hydrophobic group via a bond chain. GAG has a functional group which is a carboxyl, hydroxyl or sulfonic acid (—SO ₃ H) group as a side chain. Thus, the hydrophobic group is bonded to GAG via a bonding chain obtained by forming an ether bond, a carboxylic acid ester bond, a sulfate ester bond, a carboxylic acid amide bond or a sulfonic acid amide bond together with these functional groups. can do. Such a linking chain may specifically include —CONH—, —COO—, —O—, —SO ₃ — and —SO ₂ NH—. Among these, a —CONH— carboxylic acid amide bond and a —COO— carboxylic acid ester bond can be preferably used, and a —CONH— carboxylic acid amide bond can be particularly preferably used.

4) Spacer chain In GAG into which a hydrophobic group is introduced via a binding chain, which is contained in the agent for applying to mucosa of the present invention, the hydrophobic group is bound to GAG via the binding chain, and the spacer chain is , May be further present between the binding chain and the hydrophobic group. As such a spacer chain, any chain group can be used as long as the spacer group does not completely eliminate the pharmacological action of GAG. Specifically, — (CH ₂ ) _m — and — (CH ₂ ) — (OCH ₂ ) _n — (wherein m and n are each an integer of 1 to 18) may be included.
These spacer chains may further have a bonding chain such as —CONH—, —COO—, —O—, —SO ₃ — and —SO ₂ NH— as described above on the hydrophobic group side. Specifically, such a spacer chain having a binding chain on the hydrophobic group side includes —COO— (CH ₂ ) _m —, —COO— (CH ₂ ) — (OCH ₂ ) _n —, —CONH— (CH ₂ ) _m - and _{-CONH- (CH 2) - (OCH} 2) n - can contain.

5) Ratio having a hydrophobic group In the GAG into which a hydrophobic group is introduced via a binding chain, contained in the agent for applying to mucosa of the present invention, it is not necessary that all of the GAG structural units have a hydrophobic group. The ratio of the molar equivalents of bound hydrophobic groups to the molar equivalents of GAG disaccharide repeat units (hereinafter referred to as “introduction rate”) is the type of hydrophobic group, the required degree of hydrophobicity, the mucosal application agent. It can be appropriately determined depending on the type of mucosal disorder to be administered and the site of administration. For example, when an optionally substituted phenylethenyl group is used as the hydrophobic group, it is preferably 5 to 30%, and more preferably 10 to 20%, based on the molar equivalent of the disaccharide repeating unit of GAG. A molar equivalent of a hydrophobic group is introduced (when the below-mentioned cross-linking is not formed).

6) Crosslink-forming group In GAG in which a hydrophobic group is introduced via a bond chain, which is included in the agent for applying to mucosa of the present invention, the hydrophobic group forms a crosslink between GAG molecules by the functional group included in the group. May be. As the hydrophobic group capable of forming a cross-linked bond, any group can be used as long as the hydrophobic group causes a photodimerization reaction or a photopolymerization reaction upon irradiation with ultraviolet rays and has the same meaning as described above. Hydrophobic groups that can form crosslinks include, for example, phenylethenyl, p-aminophenylethenyl, ethenyl, 2-carboxyethenyl, and pentane-1,3-dienyl. These groups are preferably bonded to GAG via a bond chain containing a carbonyl group. Among these hydrophobic groups, phenylethenyl or p-aminophenylethenyl that is bonded to GAG via a bond chain containing a carbonyl group can be particularly preferably used.
In such GAG into which a hydrophobic group capable of forming a crosslink is introduced, GAG molecules can be cross-linked with each other by being subjected to a photodimerization reaction or a photopolymerization reaction by a conventional method. For example, it can be subjected to a photodimerization reaction or a photopolymerization reaction by a method described in Japanese Patent Application Laid-Open No. 2002-249501.

7) Preferred GAG Constituent Unit As a typical example of GAG having a hydrophobic group introduced via a binding chain, contained in the agent for applying to mucosa of the present invention, specifically, Ph—CH═CH—COO— (CH _{2 ) m -N H-; Ph-CH} = CH-COO-CH 2 - (OCH 2) n -N H-; Ph-CH = CH-CONH- (CH 2) m -N H-; Ph-CH = _{CH-CONH-CH 2 - (} OCH 2) n -N H-; Ph-CH = CH-COO- (CH 2) m - O-; Ph-CH = CH-COO-CH 2 - (OCH 2) n - O-; Ph-CH = CH -CONH- (CH 2) m - O-; Ph-CH = CH-CONH-CH 2 - (OCH 2) n - O-; CH 3 - (CH 2) l - COO—Ph—CONH— (CH ₂ ) _m —N H— or CH ₃ — (CH ₂ ) ₁ —COO—Ph— CONH—CH ₂ — (OCH ₂ ) _n —N H— (wherein Ph represents a phenyl group, m and n each represents an integer of 1 to 18 and l represents 0 or 1 to 18) And an agent for applying to mucosa containing GAG having an integer) as an active ingredient.
The following GAGs can be mentioned as typical examples.
Chemical formula 1:

[Wherein R represents R ₁ or R ₂ ;
Ac represents an acetyl group;
R ₁ represents ONa or OH;
R ₂ represents (1) Ph—CH═CH—COO— (CH ₂ ) _m —NH—;
(2) Ph—CH═CH—COO—CH ₂ — (OCH ₂ ) _n —NH—;
(3) Ph—CH═CH—CONH— (CH ₂ ) _m —NH—;
(4) Ph—CH═CH—CONH—CH ₂ — (OCH ₂ ) _n —NH—;
(5) Ph—CH═CH—COO— (CH ₂ ) _m —O—;
(6) Ph—CH═CH—COO—CH ₂ — (OCH ₂ ) _n —O—;
(7) Ph—CH═CH—CONH— (CH ₂ ) _m —O—;
(8) Ph—CH═CH—CONH—CH ₂ — (OCH ₂ ) _n —O—;
_{(9) CH 3 - (CH} 2) l -COO-Ph-CONH- (CH 2) m -NH-; or _{(10) CH 3 - (CH} 2) l -COO-Ph-CONH-CH 2 - ( OCH ₂ ) _n —NH— (wherein Ph represents a phenyl group, m and n each represent an integer of 1 to 18 and l represents 0 or an integer of 1 to 18)] GAG having a repeating structure of a structural unit represented by a basic skeleton [wherein the ratio of the molar equivalent of the structural unit in which R represents R ₂ to the molar equivalent of the disaccharide repeating unit of GAG is 5 to 30% Is].

8) Method for producing GAG in which a hydrophobic group is introduced through a binding chain In order to obtain GAG into which a hydrophobic group has been introduced through a binding chain, GAG is prepared by adding the above hydrophobic group to carboxyl, hydroxyl or sulfone in GAG. Functional groups such as hydroxyl, carboxyl, amino or sulfonic acid groups that can form an ether bond, a carboxylic ester bond, a sulfate ester bond, a carboxylic acid amide bond or a sulfonic acid amide bond with an acid (—SO ₃ H) group It reacts with a hydrophobic compound bound to the. Specifically, when this bond is a carboxylic acid amide bond, the carboxyl group in GAG is converted to an amino group in the hydrophobic compound by reacting GAG having a carboxyl group with a hydrophobic compound having an amino group. Combine. In the case of a carboxylic acid ester bond, the GAG is reacted with a hydrophobic compound having a hydroxyl or carboxyl group to bond the carboxyl group in the GAG to the hydroxyl group in the hydrophobic compound, or the hydroxyl group in the GAG. The group is bonded to a carboxyl group in the hydrophobic compound. In the case of an ether bond, the hydroxyl group in GAG is reacted with the hydroxyl group in the hydrophobic compound by reacting GAG having a hydroxyl group with a hydrophobic compound having a hydroxyl group. In the case of a sulfonate ester bond, the GAG is reacted with a hydroxyl group or a hydrophobic compound having a sulfonic acid group to bond the hydroxyl group in the GAG to the sulfonic acid group in the hydrophobic compound, or GAG The sulfonic acid group in it is bonded to the hydroxyl group in the hydrophobic compound. These reactions can be carried out by a general ordinary method, and reaction conditions can be appropriately selected by those skilled in the art.

  When a spacer chain exists between the binding chain and the hydrophobic group, the order of introducing the spacer chain and the hydrophobic group into the GAG is not particularly limited. For example, functional groups such as hydroxyl, carboxyl, amino or sulfonic acid groups that can form ether bonds, carboxylic ester bonds, sulfate ester bonds, carboxylic acid amide bonds or sulfonic acid amide bonds with functional groups in GAG. A hydrophobic compound in which a spacer compound having one end of the spacer chain is reacted with GAG, and then the other end of the spacer compound is bonded to a functional group such as hydroxyl, carboxyl, amino or sulfonic acid group Or a hydroxyl, carboxyl, amino or sulfonic acid that can form an ether bond, carboxylic ester bond, sulfate ester bond, carboxylic acid amide bond or sulfonic acid amide bond with a functional group in a hydrophobic compound Have a functional group at one end A spacer compound having a hydrophobic group bonded to a functional group such as hydroxyl, carboxyl, amino or sulfonic acid group, and then reacting the other end of the spacer compound with GAG Any of these can be used. In particular, a method in which a spacer compound is reacted with a hydrophobic compound and subsequently reacted with GAG can be preferably used.

The above method can be appropriately carried out by a known method, but is preferably carried out in the presence of a condensing agent. Such condensing agents are preferably water-soluble carbodiimides such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI.HCl), dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinic acid. Mention may be made of condensing agents such as imides (HOSu). For example, hyaluronic acid is used as GAG, and as a hydrophobic compound bound to a spacer compound, Ph—CH═CH—COO— (CH ₂ ) _m —NH ₂ or Ph—CH═CH—COO—CH ₂ — ( OCH _{2) n} -NH ₂ (where if m and n are each the use of cinnamic acid derivatives, such as a a) 1 to 18 integer, 1-ethyl-3- (3-dimethylaminopropyl) A condensation method using a water-soluble carbodiimide such as carbodiimide hydrochloride (EDCI.HCl) and N-hydroxysuccinimide can be preferably used. This reaction can be performed using a mixed solvent of water and a water-soluble organic solvent such as dioxane, dimethylformamide, or ethanol. A hyaluronic acid derivative that is highly soluble in an aqueous medium can be obtained by treatment with a base such as sodium hydrogen carbonate after completion of the reaction.

In the case where GAG molecules produced in this manner and having a hydrophobic group introduced via a bonding chain are subjected to a photodimerization reaction or a photopolymerization reaction to crosslink GAG molecules to each other, for example, see JP-A-2002-249501. The methods described can be used. Specifically, in the case of a compound in which a phenylethenyl group as a hydrophobic group is bonded to GAG via —COO— (CH ₂ ) _m —NHCO—, the crosslinking is a solution containing these using an ultraviolet lamp. Can be formed by light irradiation.

9) Mucosal application agent of the present invention The mucosal application agent of the present invention contains one or more GAGs into which a hydrophobic group is introduced via a binding chain as an active ingredient, and the hydrophobic group is also introduced via a binding chain. It may further comprise other medically, pharmacologically or biologically acceptable substances other than the prepared GAG. Such materials include, but are not limited to, salts such as sodium chloride, potassium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate and monopotassium hydrogen phosphate, and paraoxybenzoates, benzalkonium chloride Preservatives such as chlorobutanol and chlorhexidine gluconate, and other pharmacologically active ingredients.
The agent for applying to mucosa of the present invention can be applied to any known dosage form as a medicament for mucosal application (for example, solid preparations such as granules and powders, liquid preparations such as aqueous solutions, suspensions and emulsions, and gels). Formulation). In the agent for applying to mucosa of the present invention, the dosage form when formulated and distributed and the dosage form when applied to the mucous membrane may be the same or different. For example, the agent for applying to mucosa of the present invention can be formulated in a liquid dosage form and directly applied to the mucous membrane as it is. In addition, the agent for applying to mucosa of the present invention may be formulated and distributed as a solid dosage form to be a liquid or gel when applied to the mucous membrane. Therefore, the agent for applying to mucosa of the present invention can be made into a preparation for preparation at the time of use.
In the case of preparing a solution by dissolving in water, the amount of GAG having a hydrophobic group introduced via a bonding chain is preferably 0.02 to 5% by weight, more preferably 0.1 to 3% by weight, and very preferably Is 0.1 to 1 wt%, and most preferably 0.1 to 0.6 wt%.

<Applicable target>
The mucosal agent of the present invention is intended to be applied to the mucosa. The animal to which the agent for applying mucosa of the present invention is applied is not particularly limited as long as it has a mucosa, but mammals are preferable. Examples of mammals include, but are not limited to, humans, horses, cows, dogs, cats, rabbits, hamsters, guinea pigs and mice. Naturally, the agent for applying to mucosa of the present invention can be used as a medicine for humans, and can also be used as an animal medicine. Especially, it is preferable to make it a human preparation.
The mucosa to which the agent for applying mucosa of the present invention can be applied is not particularly limited as long as the mucosa is a mucosa present in animals. Such mucous membranes include the digestive system such as the stomach and intestine, the circulatory system, the respiratory system, the excretory system such as the bladder, rectum and anus, the genital system such as the vagina, and the eyes, nose and oral cavity. Examples include mucosal tissues existing in organs and tissues exemplified by organs in contact with the outside world. Among these, the mucosa application agent of the present invention can be preferably applied to the cornea, conjunctiva, oral mucosa and bladder mucosa.

<Applicable disease>
The mucosa application agent of the present invention can be applied to such mucosa in a wide range. The purpose of application is not particularly limited, and examples thereof include protection of mucosal tissues (for example, prevention of snow eyes, pterygium and cataracts by ultraviolet rays), prevention of mucosal dryness, and treatment of mucosal disorders. it can. Therefore, the agent for applying to mucosa of the present invention can be applied not only to an abnormal mucosa (for example, a mucous membrane in which a disorder has occurred) but also to a normal mucosa. However, since the agent for applying to mucosa of the present invention exerts an excellent pharmacological action in the mucosa where the disorder has occurred, it is preferably used for the treatment of mucosal disorders such as those in the cornea, conjunctiva, oral mucosa and bladder mucosa. it can.

  Since the agent for applying to mucosa of the present invention exerts a pharmacological action excellent in the damage in the mucosal epithelium among the mucosal disorders, it can be preferably used for the treatment of the disorder in the mucosal epithelium.

  Examples of such disorders in the mucosal epithelium include corneal epithelial layer disorders such as dry cornea (dry eye), keratoconjunctivitis, punctate superficial keratitis (SPK), corneal epithelial erosion, corneal epithelial defect and corneal tumor; Oral mucosal disorders such as xerostomia (dry mice), aphthous ulcers, stomatitis and glossitis; dryness and pruritus of nasal mucosa; bladder mucosal disorders such as interstitial cystitis; ulcerative proctitis and rectal or Including vaginal dryness. In addition, dryness and damage of the organ mucosa during surgery can also be mentioned. Among these, it can be preferably used for the treatment of corneal epithelial layer disorder, oral mucosal epithelial layer disorder and bladder mucosal epithelial layer disorder.

Application method and amount The agent for applying to mucosa of the present invention can be applied to the mucosal tissues exemplified above, and those skilled in the art can determine the application method and application form of the position, form of mucosa to be applied, It can be determined appropriately according to the properties and functions, and the purpose of application. However, it is preferable that the agent for applying to mucosa of the present invention is applied to the mucous membrane as a liquid agent such as a solution in use. In that case, at the time of production (formulation) or application of the agent for applying to mucosa of the present invention, this solution can be obtained by dissolving GAG into which a hydrophobic group has been introduced via a binding chain in a solvent. The solvent is not particularly limited as long as it is a pharmaceutically acceptable solvent that can dissolve GAG into which a hydrophobic group has been introduced via a binding chain. For example, a buffer solution such as a phosphate buffer solution or physiological saline can be used, but the solvent is not limited thereto. In this case, the concentration of GAG into which a hydrophobic group is introduced via a binding chain in the liquid preparation is not particularly limited, and can be appropriately determined according to the type of mucosa to be applied and the degree of mucosal damage. When the mucosa application agent of the present invention is an eye drop, when the mucosa application agent of the present invention is applied to the oral mucosa or bladder mucosa, for example, the concentration is preferably 0.02 to 5% by weight, more preferably 0. 0.1 to 3% by weight, more preferably 0.1 to 1% by weight, still more preferably 0.1 to 0.6% by weight, very preferably 0.1 to 0.5% by weight, and most preferably 0.1 to 0.3% by weight.

  When applying the mucosal application agent of the present invention to the gastric mucosa as a liquid as described above, an oral administration or administration method using a catheter can be selected. For example, when it is applied to the mucous membrane of the eye, nose or mouth, an administration method such as eye drops, nose drops or gargle can be selected. For example, when the agent for applying mucosa of the present invention is applied to the bladder, rectum or vaginal mucosa, or the mucous membrane of an organ whose dryness is related to surgery, the agent for applying mucosa of the present invention to the lumen of these organs or tissues or Methods of administration can be selected by injection, spraying or application to the surface, but are not limited to these methods.

  The amount (frequency) and frequency of application (administration) of the agent for applying to mucosa of the present invention are not particularly limited, but the mucosa to be applied, the purpose of application, the type of animal to be applied, age, weight, sex, and mucosal disorder It should be decided according to the degree.

  Specifically, when the agent for applying to mucosa of the present invention is used for the purpose of treating a human corneal epithelial layer disorder, a liquid preparation for eye drops containing GAG having a hydrophobic group introduced via a binding chain at the above-mentioned concentration. The agent for applying to mucosa of the present invention as (eye drops) can be administered by instilling 1 to 3 drops per administration, 1 to 5 times a day, and 1 to 3 drops per administration. It may be administered by instilling 1 to 3 times a day.

  When the mucosal application agent of the present invention is used for the purpose of treating human oral mucosal disorders, the mucosa application agent of the present invention as a solution containing GAG having a hydrophobic group introduced through a binding chain at the above-mentioned concentration is as follows. The mucosal application agent of the present invention can be administered by including in the oral cavity 1 to 5 times a day, rinsing for about several tens of seconds (preferably about 20 to 30 seconds), and then exhaling it.

  When the agent for applying mucosa of the present invention is applied to bladder mucosal disorder, interstitial cystitis, eosinophilic bladder which preferably exhibits symptoms similar to those of acute bacterial cystitis but does not react with antibacterial agents Used for the treatment of bladder mucosal disorders exemplified by non-bacterial refractory cystitis exemplified by inflammation and hemorrhagic cystitis. In this case, the mucosa application agent of the present invention as a solution containing GAG having a hydrophobic group introduced through a binding chain at the above-mentioned concentration is 1 to 7 times a week in an amount of 50 mL per administration. It can be administered by directly administering the agent for applying mucosa of the present invention to the bladder or by administering it to the bladder with a catheter.

  The agent for applying to mucosa of the present invention has a long retention in the affected area because the active ingredient contained in this agent exhibits a higher retention in the mucosa compared to conventional drugs containing hyaluronic acid to which hydrophobic groups are not bound as an active ingredient. You can stay for hours. Therefore, the agent for applying to mucosa of the present invention can also continuously exert a therapeutic effect against disorders such as inflammation and damage of the mucous membrane even at a low administration frequency. However, the agent for applying to mucosa of the present invention is not limited by the administration frequency.

Hereinafter, the present invention will be described by way of examples.
<Example 1>
(1-1) Preparation of cinnamic acid derivative-introduced sodium hyaluronate 172 mg / 5 mL of an aqueous solution of N-hydroxysuccinimide (HOSu: Watanabe Chemical Co., Ltd.), 143 mg / 5 mL of 1-ethyl-3- (3-dimethyl) Aminopropyl) carbodiimide hydrochloride (EDCI · HCl) (Watanabe Chemical Co., Ltd.) and 181 mg / 5 mL of cinnamic acid 3-aminopropyl hydrochloride (Tokyo Chemical Industry Co., Ltd.) in water (115 mL) / It was added to a solution of sodium hyaluronate (1.06 g, 2.7 mmol / disaccharide unit, weight average molecular weight 900,000; derived from chicken crown, Seikagaku Corporation) in dioxane (144 mL). The mixture was stirred for 3 hours, and 750 mg / 10 mL of an aqueous solution of sodium bicarbonate (Japanese Pharmacopoeia) was added. After further stirring for 2 hours and 30 minutes, the reaction was stopped with acetic acid (214 mg) and sodium chloride (1.0 g). Ethanol (300 mL) was added, and the resulting precipitate was filtered off and washed twice with 80% ethanol and 95% ethanol in this order. This solid was dried overnight at 40 ° C. under vacuum to obtain a white solid (1.06 g) (hereinafter, “cinnamate derivative-introduced sodium hyaluronate” was referred to as “cinnamate derivative-introduced HA”). For short). The introduction rate of the cinnamic acid derivative was 16%. The introduction rate of the cinnamic acid derivative was calculated based on the amount of the cinnamic acid derivative by the absorbance measurement method (wavelength: 269 nm) and the amount of hyaluronic acid by the carbazole sulfate method.
(1-2) Preparation of cinnamic acid derivative-introduced HA solution By adding physiological saline to 86 mg of cinnamic acid derivative-introduced HA obtained in (1-1) above, the total amount is 15.45 ml, and then uniformly The solution was shaken on a shaker overnight until dissolved. A 0.5% by weight solution of cinnamic acid derivative-introduced HA (loss on drying: 10%) was obtained. Similarly, 0.3 wt% and 0.1 wt% solutions of cinnamic acid derivative-introduced HA were obtained.

<Example 2>
(2-1) Preparation of cinnamic acid derivative-introduced HA 75 mg / 5 mL HOSu aqueous solution, 62 mg / 5 mL EDCI · HCl water for injection (hereinafter referred to as WFI), and 92 mg / 5 mL cinnamon WFI solution of acid 6-aminohexyl hydrochloride (Tokyo Chemical Industry Co., Ltd.) was added to sodium hyaluronate (1.0 g, 2.5 mmol / disaccharide unit, weight average molecular weight 1 in WFI (150 mL) / dioxane (75 mL). , 500,000; derived from chicken crown, Seikagaku Corporation). The mixture was stirred for 4 hours and sodium chloride (1.0 g) was added. Ethanol (500 mL) was added, and the resulting precipitate was filtered off and washed twice with 80% ethanol and ethanol in this order. The solid was dried at 40 ° C. in vacuo to give a white solid (1.1 g). The introduction rate of the cinnamic acid derivative was 2.7%.
(2-2) Preparation of crosslinked cinnamic acid derivative-introduced HA The above cinnamic acid derivative-introduced HA (12.5 g) was converted into phosphate buffered saline (phosphate concentration: 1.5 mM, hereinafter referred to as “PBS A 2.5% solution of cinnamic acid derivative-introduced HA was prepared (500 mL). The 2.5% solution of the cinnamic acid derivative-introduced HA was irradiated with an 800 W high-pressure mercury lamp and heat-treated in an autoclave at 121 ° C. for 7.5 minutes to obtain a crosslinked cinnamic acid derivative-introduced HA.
Further, 1 g of the crosslinked cinnamic acid derivative-introduced HA was dissolved in 11.5 ml of WFI to prepare a 0.2 wt% solution of the crosslinked cinnamic acid derivative-introduced HA.

<Example 3>
(3-1) Preparation of Cinnamic Acid Derivative-Introduced HA 172 mg / 5 mL HOSu aqueous solution, 143 mg / 5 mL EDCI · HCl aqueous solution, and 181 mg / 5 mL cinnamic acid 3-aminopropyl hydrochloride (Tokyo Chemical Industry Co., Ltd.) Company) aqueous solution of sodium hyaluronate (1.0 g, 2.5 mmol / disaccharide unit, weight average molecular weight 900,000; derived from chicken crown, Seikagaku Corporation) in water (150 mL) / dioxane (75 mL) Added to the solution. The mixture was stirred for 3 hours and an aqueous solution of 750 mg / 10 mL of sodium bicarbonate (Japanese Pharmacopoeia) was added. After further stirring for 2 hours and 30 minutes, the reaction was quenched with acetic acid (214 mg) and sodium chloride (1.0 g). Ethanol (300 mL) was added, and the resulting precipitate was filtered off and washed twice with 80% ethanol and 95% ethanol in this order. This solid was dried at 40 ° C. under vacuum to obtain a white solid (1.0 g) as cinnamic acid derivative-introduced HA. The introduction rate of the cinnamic acid derivative was 10.1%.
(3-2) Preparation of fluorescent dye-labeled cinnamic acid derivative-introduced HA 3.0 mmol / mL HOSu aqueous solution, 1.5 mmol / mL EDCI · HCl aqueous solution and 1.5 mmol / mL 4-aminofluorescein (Tokyo) Kasei Kogyo Co., Ltd.) was added to the cinnamic acid derivative-introduced HA (1.00 g, 2.5 mmol / disaccharide unit) obtained in (3-1) above in water (150 mL) / dioxane (75 mL). Added to the solution. The mixture was stirred for 1 day and an aqueous solution of 500 mg / 10 mL of sodium bicarbonate (Japanese Pharmacopoeia) was added. After further stirring for 4 hours and 30 minutes, the reaction was quenched with acetic acid (2 mL) and sodium chloride (6.0 g). Ethanol (500 mL) was added and the resulting precipitate was filtered off and washed 4 times with 80% ethanol and 2 times with ethanol. This solid was dried in vacuum overnight to obtain a fluorescent dye-labeled solid (782 mg). The introduction rate of fluorescence was 0.60%.

<Comparative Example 1>
Preparation of fluorescent dye-labeled HA An aqueous solution of 3.0 mmol / mL HOSu, an aqueous solution of 1.5 mmol / mL EDCI · HCl, and an aqueous solution of 1.5 mmol / mL 4-aminofluorescein (Tokyo Chemical Industry Co., Ltd.) (150 mL) / dioxane (75 mL) in sodium hyaluronate (1.00 g, 2.5 mmol / disaccharide unit, weight average molecular weight 900,000; derived from chicken crown, Seikagaku Corporation). The mixture was stirred for 1 day and an aqueous solution of 500 mg / 10 mL of sodium bicarbonate (Japanese Pharmacopoeia) was added. After further stirring for 4 hours and 30 minutes, the reaction was quenched with acetic acid (2 mL) and sodium chloride (6.0 g). Ethanol (500 mL) was added and the resulting precipitate was filtered off and washed 4 times with 80% ethanol and 2 times with ethanol. This solid was dried in a vacuum overnight to obtain a fluorescent dye-labeled solid (830 mg). The introduction rate of fluorescence was 0.32%.

<Example 4>
Measurement of UV transmittance of cinnamic acid derivative-introduced HA solution An aqueous solution of cinnamic acid derivative-introduced HA obtained in (1-1) of 0.1% by weight was prepared, and a spectrometer (UV-1600, stock) UV transmittance was measured by Shimadzu Corporation.
The spectrum showing the transmittance is shown in FIG. 1, and the transmittance (%) at various wavelengths is shown in Table 1. As a result, the transmittance of 100% was shown at a wavelength of 340 nm or more, but the transmittance at a wavelength of about 320 nm or less is as extremely low as 20% or less, so this solution effectively blocks ultraviolet light transmission. It was proved.
In FIG. 1, the scales are shown with 65 nm intervals on the horizontal axis and 20% intervals on the vertical axis.

In the table, λ and T represent wavelength and transmittance (%), respectively.

<Example 5>
Effect of cinnamate derivative-introduced HA on healing of rabbit corneal epithelium Effect of cinnamate derivative-introduced HA prepared in Example 1 on the healing of surgically excised rabbit corneal epithelium (surgical model).
(5-1) Method 1) Surgical resection of corneal epithelium (surgical model)
After anesthesia by intravenous injection of 5 mg / kg ketamine and 2 mg / kg xylazine and topical administration of 0.4% oxybuprocaine hydrochloride, the central part of the corneal epithelium was placed with trephine (inner diameter 8 mm), 23G needle and It was excised by using a micro scissor.
2) Topical administration 1 hour and 4 hours after peeling the corneal epithelium, 150 μl of physiological saline was administered to the left eye as a control substance, and 0.5% by weight of cinnamon prepared in Example (1-2) above 150 μl of acid derivative-introduced HA solution was administered to the right eye as a test substance. One and two days after exfoliation, the same administration as described above was performed a total of 4 times at 3 hour intervals and 3 days after exfoliation at 3 hour intervals. For administration, a 1 ml injection syringe was used. Six model rabbits with corneal epithelial layer disorder described in 1) above were used as administration subjects.
3) Imaging of corneal epithelial defect Rabbits were given general anesthesia by intravenous injection of 5 mg / kg ketamine and 2 mg / kg xylazine, and then the corneal epithelial disappearance site was treated with 0.2% sodium fluorescein dissolved in PBS. Stained and photographed under UV light. Photographing was performed immediately before administration of the test substance 1 hour after the cornea was detached, and 3 hours after the final administration 3 days after the separation. At the time of shooting, the focal length was fixed so that the magnification of the photograph was constant.
4) Measurement of corneal epithelial defect The area of the corneal epithelial defect stained with fluorescein sodium was measured on the printed photograph using an image analyzer. By subtracting the area of the exfoliation site 3 hours after the final administration 3 days after exfoliation from the area (exfoliation area) of the exfoliation site immediately before administration of the test substance 1 hour after exfoliation of the corneal epithelium. The obtained value was defined as “healing area”.

(5-2) Test Results The results of the healing area rate of each individual are shown in FIG. 2, and the results of the healing area rate and the healing area rate ratio of each individual are shown in Table 2. The healing area rate and the healing area rate ratio were calculated as follows.
Healing area rate (%) = (healing area / peeling area) × 100
Healing area rate ratio = (Healing area rate of the right eye / Healing area rate of the left eye) × 100

  For each individual of individual numbers 1 to 6, the left column shows the healing area rate of the right eye (cinnamic acid derivative-introduced HA administration), and the right column shows the healing area of the left eye (saline administration) Indicates the rate. In FIG. 2 and Table 2, the obvious effect of promoting the healing of corneal epithelial layer disorder was observed in 5 out of 6 administered individuals.

<Example 6>
Effect of 0.5% cinnamate derivative-introduced HA on healing of rabbit corneal epithelium Effect of cinnamic acid derivative-introduced HA prepared in Example 1 on healing of surgically excised rabbit corneal epithelium (surgical model) ).
(6-1) Method 1) Surgical resection of corneal epithelium (surgical model)
After anesthesia by intravenous injection of 5 mg / kg ketamine and 2 mg / kg xylazine and topical administration of 0.4% oxybuprocaine hydrochloride, the central part of the corneal epithelium was placed with trephine (inner diameter 8 mm), 23G needle and It was excised by using a micro scissor.
2) Topical administration 1 hour and 4 hours after peeling the corneal epithelium, 150 μl of physiological saline was administered to the left eye as a control substance, and 0.5% by weight of cinnamon prepared in Example (1-2) above 150 μl of acid derivative-introduced HA solution was administered to the right eye as a test substance. One and two days after exfoliation, the same administration as described above was performed a total of 4 times at 3 hour intervals and 3 days after exfoliation at 3 hour intervals. For administration, a 1 ml injection syringe was used. Fourteen model rabbits with corneal epithelial layer disorder described in 1) above were used as administration subjects.
3) Imaging of corneal epithelial defect Rabbits were given general anesthesia by intravenous injection of 5 mg / kg ketamine and 2 mg / kg xylazine, and then the corneal epithelial disappearance site was treated with 0.2% sodium fluorescein dissolved in PBS. Stained and photographed under UV light. Photographing was performed immediately before administration of the test substance 1 hour after exfoliating the cornea and 3 hours after the second administration 1 to 3 days after exfoliation. At the time of shooting, the focal length was fixed so that the magnification of the photograph was constant.
4) Measurement of corneal epithelial defect The area of the corneal epithelial defect stained with fluorescein sodium was measured on the printed photograph using an image analyzer. It is obtained by subtracting the area of the exfoliation site 3 hours after the final administration 3 days after exfoliation from the area (exfoliation area) of the exfoliation site immediately before administration of the test substance 1 hour after exfoliation of the corneal epithelium. The value was defined as “healing area”.

(6-2) Test Results The results of the healing area of each individual are shown in FIG. 3, and the results of the healing rate of each individual are shown in FIG. The healing area and healing rate were calculated as follows.
Healing area = Area after exfoliation ^* -Area at each time point (after 1 to 3 days)
* In the present specification, “area after exfoliation” means “immediately before exfoliation of the test substance 1 hour after exfoliating the cornea” in calculating the healing area.
Healing rate = average value of healing area at each time point (after 1 to 3 days) In FIGS. 3 and 4, the healing area of the corneal epithelium is 0.5% by weight of cinnamon compared with the healing area of the corneal epithelium of the control eye It was observed that there was a significant increase in eyes administered with acid derivative-introduced HA solution. It was also observed that the healing rate was significantly increased in the eyes administered with 0.5 wt% cinnamic acid derivative-introduced HA solution.

<Example 7>
Effect of 0.3% cinnamic acid derivative-introduced HA on healing of rabbit corneal epithelium Effect of cinnamic acid derivative-introduced HA prepared in Example 1 on healing of surgically excised rabbit corneal epithelium (surgical model) ).
(7-1) Method 1) Surgical resection of corneal epithelium (surgical model)
After anesthesia by intravenous injection of 5 mg / kg ketamine and 2 mg / kg xylazine and topical administration of 0.4% oxybuprocaine hydrochloride, the central part of the corneal epithelium was placed with trephine (inner diameter 8 mm), 23G needle and It was excised by using a micro scissor.
2) Topical administration 1 hour and 4 hours after the corneal epithelium was detached, 150 μl of physiological saline was administered to the left eye as a control substance, and 0.3% by weight of cinnamon prepared in Example (1-2) above 150 μl of acid derivative-introduced HA solution was administered to the right eye as a test substance. One and two days after exfoliation, the same administration as described above was performed a total of 4 times at 3 hour intervals and 3 days after exfoliation at 3 hour intervals. For administration, a 1 ml injection syringe was used. Fourteen model rabbits with corneal epithelial layer disorder described in 1) above were used as administration subjects.
3) Imaging of corneal epithelial defect Rabbits were given general anesthesia by intravenous injection of 5 mg / kg ketamine and 2 mg / kg xylazine, and then the corneal epithelial disappearance site was treated with 0.2% sodium fluorescein dissolved in PBS. Stained and photographed under UV light. Photographing was performed immediately before administration of the test substance 1 hour after exfoliating the cornea and 3 hours after the second administration 1 to 3 days after exfoliation. At the time of shooting, the focal length was fixed so that the magnification of the photograph was constant.
4) Measurement of corneal epithelial defect The area of the corneal epithelial defect stained with fluorescein sodium was measured on the printed photograph using an image analyzer. It is obtained by subtracting the area of the exfoliation site 3 hours after the final administration 3 days after exfoliation from the area (exfoliation area) of the exfoliation site immediately before administration of the test substance 1 hour after exfoliation of the corneal epithelium. The value was defined as “healing area”.

(7-2) Test Results The results of the healing area of each individual are shown in FIG. 5, and the results of the healing rate of each individual are shown in FIG. The healing area and healing rate were calculated as follows.
Healing area = Area after exfoliation−Area at each time point (after 1 to 3 days) Healing rate = Average value of the healing area at each time point (after 1 to 3 days) In FIGS. Compared with the healing area of the corneal epithelium, it was observed that the eye administered with 0.3 wt% cinnamic acid derivative-introduced HA solution significantly increased at all time points on the 1st to 3rd days. It was also observed that the healing rate was significantly increased in the eyes administered with 0.3 wt% cinnamic acid derivative-introduced HA solution.

<Example 8>
The effect of 0.1% cinnamic acid derivative-introduced HA on the healing of rabbit corneal epithelium (0.1% by weight cinnamic acid derivative-introduced HA aqueous solution and 0.1% by weight HA aqueous solution, instilled 4 times a day)
The effect of the cinnamic acid derivative-introduced HA prepared in Example 1 on the healing of surgically excised rabbit corneal epithelium (surgical model).
(8-1) Method 1) Surgical resection of corneal epithelium (surgical model)
After anesthesia by intravenous injection of 5 mg / kg ketamine and 2 mg / kg xylazine and topical administration of 0.4% oxybuprocaine hydrochloride, the central part of the corneal epithelium was placed with trephine (inner diameter 8 mm), 23G needle and It was excised by using a micro scissor.
2) Topical administration 1 hour and 4 hours after the corneal epithelium was detached, 150 μl of 0.1 wt% aqueous solution of HA having a weight average molecular weight of 600,000 to 1,200,000 was administered to the left eye as a control substance, and the above 150 μl of 0.1 wt% cinnamic acid derivative-introduced HA solution prepared in Example (1-2) was administered as a test substance to the right eye. One and two days after exfoliation, the same administration as described above was performed a total of 4 times at 3 hour intervals and 3 days after exfoliation at 3 hour intervals. For administration, a 1 ml injection syringe was used. Eight model rabbits with corneal epithelial layer disorder described in 1) above were used as administration subjects.
3) Imaging of corneal epithelial defect Rabbits were given general anesthesia by intravenous injection of 5 mg / kg ketamine and 2 mg / kg xylazine, and then the corneal epithelial disappearance site was treated with 0.2% sodium fluorescein dissolved in PBS. Stained and photographed under UV light. Photographing was performed immediately before administration of the test substance 1 hour after exfoliating the cornea and 3 hours after the second administration 1 to 3 days after exfoliation. At the time of shooting, the focal length was fixed so that the magnification of the photograph was constant.
4) Measurement of corneal epithelial defect The area of the corneal epithelial defect stained with fluorescein sodium was measured on the printed photograph using an image analyzer. It is obtained by subtracting the area of the exfoliation site 3 hours after the final administration 3 days after exfoliation from the area (exfoliation area) of the exfoliation site immediately before administration of the test substance 1 hour after exfoliation of the corneal epithelium. The value was defined as “healing area”.

(8-2) Test results The results of the healing area of each individual are shown in FIG. 7, and the results of the healing area and healing rate of each individual are shown in FIG. The healing area and healing rate were calculated as follows.
Healing area = Area after exfoliation−Area at each time point (after 1 to 3 days) Healing rate = Average value of the healing area at each time point (after 1 to 3 days) In FIGS. A significant increase was observed in eyes administered with 0.1 wt% cinnamic acid derivative-introduced HA solution compared to the healing area of the corneal epithelium of control eyes administered with wt% HA aqueous solution. It was also observed that the healing rate was significantly increased in the eyes administered with 0.1 wt% cinnamic acid derivative-introduced HA solution.

<Example 9>
The effect of 0.1% cinnamic acid derivative-introduced HA on the healing of rabbit corneal epithelium (0.1% by weight cinnamic acid derivative-introduced HA aqueous solution and 0.1% by weight HA aqueous solution, instilled once a day)
The effect of the cinnamic acid derivative-introduced HA prepared in Example 1 on the healing of surgically excised rabbit corneal epithelium (surgical model).
(9-1) Method 1) Surgical resection of corneal epithelium (surgical model)
After anesthesia by intravenous injection of 5 mg / kg ketamine and 2 mg / kg xylazine and topical administration of 0.4% oxybuprocaine hydrochloride, the central part of the corneal epithelium was placed with trephine (inner diameter 8 mm), 23G needle and It was excised by using a micro scissor.
2) Topical administration One hour after the corneal epithelium was detached, 150 μl of a 0.1% by weight aqueous solution of HA having a weight average molecular weight of 600,000 to 1,200,000 was administered to the left eye as a control substance. 150 μl of 0.1 wt% cinnamic acid derivative-introduced HA solution prepared in 1-2) was administered as a test substance to the right eye. Furthermore, the same administration as described above was carried out once a day 1 to 3 days after peeling. For administration, a 1 ml injection syringe was used. Eight model rabbits with corneal epithelial layer disorder described in 1) above were used as administration subjects.
3) Imaging of corneal epithelial defect Rabbits were given general anesthesia by intravenous injection of 5 mg / kg ketamine and 2 mg / kg xylazine, and then the corneal epithelial disappearance site was treated with 0.2% sodium fluorescein dissolved in PBS. Stained and photographed under UV light. Photographing was performed immediately before administration of the test substance 1 hour after peeling the cornea and 6 hours after administration 1 to 3 days after peeling. At the time of shooting, the focal length was fixed so that the magnification of the photograph was constant.
4) Measurement of corneal epithelial defect The area of the corneal epithelial defect stained with fluorescein sodium was measured on the printed photograph using an image analyzer. It is obtained by subtracting the area of the exfoliation site 3 hours after the final administration 3 days after exfoliation from the area (exfoliation area) of the exfoliation site immediately before administration of the test substance 1 hour after exfoliation of the corneal epithelium. The value was defined as “healing area”.

(9-2) Results The results of the healing area of each individual are shown in FIG. 9, and the results of the healing rate of each individual are shown in FIG. The healing area and healing rate were calculated as follows.
Healing area = Area after exfoliation−Area at each time point (after 1 to 3 days) Healing rate = Average value of the healing area at each time point (after 1 to 3 days) In FIGS. 9 and 10, the healing area of the corneal epithelium is 0.1 Compared with the healing area of the corneal epithelium of the control eye administered with the weight% HA aqueous solution, the eye administered with the 0.1 weight% cinnamic acid derivative-introduced HA solution significantly increased at all time points on the first to third days. It was observed that It was also observed that the healing rate was significantly increased in the eyes administered with 0.1 wt% cinnamic acid derivative-introduced HA solution.

<Example 10>
Fluorescence-labeled cinnamate derivative-introduced HA persistence at the corneal epithelial exfoliation site of rabbits The effect of the surgically excised rabbit corneal epithelium on the persistence of the fluorescence-labeled cinnamate derivative-introduced HA prepared in Example 3 and comparison Effect of fluorescently labeled HA prepared in Example 1 (surgical model).
(10-1) Method 1) Surgical resection of corneal epithelium (surgical model)
After anesthesia by intravenous injection of 5 mg / kg ketamine and 2 mg / kg xylazine and topical administration of 0.4% oxybuprocaine hydrochloride, the central part of the corneal epithelium was placed with trephine (inner diameter 8 mm), 23G needle and It was excised by using a micro scissor.
2) Topical administration One hour after peeling the corneal epithelium, 150 μl of a 0.3% by weight aqueous solution of fluorescently labeled HA prepared in Comparative Example 1 was administered to the left eye as a control substance, and prepared in Example 3 above. 150 μl of a 0.3% by weight aqueous solution of fluorescently labeled cinnamic acid derivative-introduced HA was administered to the right eye as a test substance. For administration, a 1 ml injection syringe was used. Eight model rabbits with corneal epithelial layer disorder described in 1) above were used as administration subjects.
3) Extraction of corneal epithelium and preparation of frozen block 30 minutes, 1 hour, 1.5 hours and 2 hours 30 minutes after administration of the test substance and the control substance, 2 rabbits, 5 mg / per rabbit General anesthesia was performed by intravenous injection of kg ketamine and 2 mg / kg xylazine, and the eyeball was removed. Using a scalpel, a hole was made between the cornea and sclera of the removed eyeball, and only the cornea was removed using a micro scissor. The extracted cornea is placed on a biological specimen chopping board (manufactured by Nissin EM Co., Ltd., catalog number 428), and the portion to be observed is a stainless steel razor (GEM (registered trademark) stainless steel uncoated) for single-edged trimming ( STAINLESS STEEL UNCOATED), Nissin EM Co., Ltd., catalog number 429). A cryostat filled with the OCT compound was soaked in an OCT compound (Tissue-Tek (registered trademark) 4583, lot 1178) and the portion to be observed next became the bottom. An unfixed frozen block was prepared by embedding in a tray (Murakaku Kogyo Co., Ltd., catalog number 31) and rapidly freezing in expanded polystyrene using liquid nitrogen.
4) Preparation of frozen section Next, the frozen block was removed from the cryostat tray and attached to the sample stage using the OCT compound. A sample stage and a microtome replacement blade (manufactured by Leica Microsystems, model 818, lot number 913212) were mounted on a high-performance freezing microtome for research, freezing chamber temperature (CT) -20 ° C. and sample side temperature (OT) -16 ° C. By cutting the block into thin sections under the conditions described above, a 5 μm-thick section was prepared using a silane-coated slide glass (manufactured by Muto Chemical Co., Ltd., Star Frost Slide Glass, catalog number 5116).
5) Observation and imaging method The frozen section was mounted on an epi-illumination fluorescence microscope (Olympus Corporation, BH2-RFC), and the FA image and autofluorescence image were respectively converted to IB cube (BH2-DMIB, excitation wavelength: 495 nm, absorption wavelength). : 460 nm) and U-cube (BH2-DMU, broadband U excitation, absorption wavelength: 435 nm). FA images and autofluorescence images were taken using a cooled high sensitivity CCD camera (Keyence Corporation, VB-6010) under conditions of an exposure time of 1 second and an ISO sensitivity of 200.

(10-2) Test results Photographs of the cornea of the specimen are shown in FIGS. From FIGS. 11 and 12, it was confirmed that the 0.3% by weight aqueous solution of the fluorescently labeled HA as a control substance remained until 30 minutes after administration but did not remain after 1 hour by the color development of the fluorescent label. confirmed. On the other hand, the fluorescent color development of the 0.3% by weight aqueous solution of the fluorescently labeled cinnamic acid derivative-introduced HA, which is the test substance, weakened with time, but at all times from 30 minutes to 2 hours 30 minutes after administration. This confirmed a high residual performance.

<Example 11>
Effect of 0.3% cinnamic acid derivative-introduced HA on the eyes of rabbits after exposure to ultraviolet rays Protective action of cinnamic acid derivative-introduced HA prepared in Example 1 on rabbits with corneal surface damage (11- 1) Test procedure 1) Rabbit anesthesia and eye opening Rabbits were anesthetized by intravenous injection of 5 mg / kg ketamine and 2 mg / kg xylazine and maintained by an inhalation of isoflurane. Next, the eyelids were always opened using a pediatric open device.
2) Administration of test substance and control substance With the eye open, 150 μl of a 0.3 wt% aqueous solution of HA having a weight average molecular weight of 600,000 to 1,200,000 is administered to the right eye as a control substance. 150 μl of 0.3 wt% cinnamic acid derivative-introduced HA prepared in Example (1-2) was administered to the left eye as a test substance. For administration, a 1 ml injection syringe was used. One rabbit described in 1) above was used as an administration subject.
3) Irradiation of ultraviolet rays to the rabbit cornea Ultraviolet rays were irradiated to both eyes using a 15 kW germicidal lamp from a distance of about 10 cm from the rabbit eyeball. Irradiation was carried out for 3 hours.
4) Imaging of the UV-irradiated site The eyeball was stained with 0.2% sodium fluorescein under continuous anesthesia of the rabbit and photographed under a purple light. At the time of shooting, the focal length was fixed so that the magnification of the photograph was constant.

(11-2) Test result The photograph after the ultraviolet irradiation is shown in FIG. From FIG. 13, in the eyeball irradiated with ultraviolet rays after administration of the control substance, the lesion site stained with 0.2% sodium fluorescein was apparent. On the other hand, in the eyeball irradiated with ultraviolet rays after administration of the test substance, the damaged site stained with 0.2% sodium fluorescein was clearly smaller than that of the control substance, and corneal damage caused by ultraviolet rays was prevented.

<Example 12>
Moisturizing effect using the isolated cornea Using the rabbit isolated cornea, the moisturizing performance of the cinnamic acid derivative-introduced HA prepared in Example 1 was verified.
(12-1) Method 1) Removal of cornea Rabbits were anesthetized by intravenous injection of 5 mg / kg ketamine and 2 mg / kg xylazine, and the eyeballs were removed. Using a scalpel, a hole was made between the cornea and sclera of the removed eyeball, and only the cornea was removed using a micro scissor.
2) Drying treatment of cornea The extracted cornea was placed on a paraffin block with a denture stabilizer and dried by supplying cold air from a distance of about 1 m from the cornea for 5 minutes using a dryer.
3) Administration of test substances (test substance, control substance and negative control substance) After completion of the drying treatment, physiological saline as a negative control substance and HA having a weight average molecular weight of 600,000 to 1,200,000 as a control substance 100 μl of a 0.3 wt% aqueous solution or a 0.5 wt% aqueous solution of cinnamic acid derivative-introduced HA prepared in Example (1-2) as a test substance was administered to two corneas. For administration, a 1 ml injection syringe was used. Three rabbits (six corneas) described in 1) above were used as administration subjects.
4) Measurement of water evaporation amount Water evaporation amount was measured using a water evaporation amount measuring device (AS-TW2, Asahi Biomed) before test substance administration, after drying treatment, after test substance administration, and test compound. Measurement was carried out at intervals of 10 minutes after administration until 40 minutes.

(12-2) Test results The measurement results of the amount of evaporated water are shown in FIG. From FIG. 14, the amount of water evaporation was slightly higher in the aqueous HA solution of the control substance than in the negative control physiological saline, but the physiological saline was close to 0 after 40 minutes. On the other hand, the amount of water evaporated after administration of the test substance remained high even after 40 minutes had elapsed, thereby confirming the clear moisture retention performance of the test substance.

<Example 13>
(13-1) Method 1) Removal of corneal epithelium The rabbit was euthanized, the eyeball was removed, and then the entire cornea was removed by incision along the sclera. The excised cornea was stored in physiological saline, and fixed by placing the corneal epithelium on a paraffin block with a dental stabilizer just before the measurement (hereinafter referred to as “the cornea to be measured”).
2) Water Evaporation of Corneal Epithelium The cornea to be measured was exposed to cold air for 5 minutes from a distance of 30 cm with a dryer and left at room temperature for 1 hour.
3) Administration of test substance After evaporation of water, physiological saline as a negative control substance, 0.3 wt% aqueous solution of HA having a weight average molecular weight of 600,000 to 1,200,000 as a control substance, or as a test substance Two drops (about 100 μl) of a 0.5% by weight aqueous solution of cinnamic acid derivative-introduced HA prepared in Example (1-2) above were administered by 1 ml syringe.
4) Measurement of water evaporation amount Using the water evaporation amount measuring device (AS-TW2), the amount perceived as undetected evaporation amount (water release amount per 1 m ² per hour) is evaporated from the cornea to be measured. Measured directly as quantity.

(13-2) Test results The measurement results of the amount of evaporated water are shown in FIG. From FIG. 15, the amount of water evaporation was slightly higher in the aqueous HA solution of the control substance than in the negative control physiological saline, but the physiological saline showed a value close to 0 after 40 minutes. On the other hand, the amount of water evaporation in the case of administration of the test substance remained high even after 40 minutes, but this caused the test substance to remain on the cornea, which is more persistent than physiological saline and aqueous HA solution. It was confirmed to have water retention.

<Example 14>
Verification of Curing Effect of Crosslinked Cinnamic Acid Derivative-Introduced HA Using a model hamster for xerostomia, the healing effect of the crosslinked cinnamic acid derivative-introduced HA prepared in Example 2 on xerostomia was verified.
(14-1) Test method 1) Preparation of model hamster for xerostomia By inserting a test tube with a diameter of about 10 mm near the buccal side in the oral cavity and turning it over under anesthesia with Nembutal, The inside of the oral cavity of Syrian hamsters was exposed. A dry hamster model hamster was obtained by applying hot air for about 20 seconds and then applying cold air for 2 minutes and 40 seconds using a drier inside the exposed oral cavity. The inside of the oral cavity was continuously exposed until the measurement was completed.
2) Administration of test substance and control substance Immediately after preparation of the xerostomia model, (A) PBS, (B) 0.2 wt% HA solution (manufactured by Seikagaku Corporation, weight average molecular weight: 1,500,000) ) Or (C) 100 μl each of 0.2 wt% crosslinked cinnamic acid derivative-introduced HA solution was applied to the inside of the oral cavity using a microsyringe.
Hereinafter, the group to which (A) is administered, the group to which (B) is administered, and the group to which (C) is administered are referred to as a PBS group, an HA group, and a crosslinked cinnamic acid derivative-introduced HA group, respectively. Regarding the administration category (administration group configuration), 7 hamsters were used for each of the PBS group, the HA group, and the HA group introduced with the crosslinked cinnamic acid derivative.
3) Calculation of water evaporation ratio The amount of water evaporation inside the oral cavity of the xerostomia model hamster is measured using the water evaporation system (Asahi Biomed), and the measured value immediately after creation of the xerostomia model hamster is 1 The water evaporation amount ratio was calculated. The greater the moisture evaporation ratio, the more moisturized state is maintained (the degree of dry mouth is low). The measurement was carried out immediately after creation of a dry mouth model hamster, immediately after administration, and 10 minutes and 20 minutes after administration.

(14-2) Test result The measurement result of the water evaporation was shown in FIG. In the figure, circle numbers 1, 2, 3 and 4 on the horizontal axis represent data immediately after preparation of xerostomia model hamster, immediately after administration, 10 minutes after administration and 20 minutes after administration, respectively. P represents a significance level. Immediately after administration, all administration groups showed a water evaporation ratio of 3.3-4.5. The water evaporation ratio 10 minutes after administration was 0.9 in the PBS group and 2.3 in the HA group. On the other hand, the average value of the crosslinked cinnamic acid derivative-introduced HA group was 3.2, indicating a higher water evaporation ratio than the PBS group and the HA group. Furthermore, the water evaporation ratio 20 minutes after administration was 0.9 in the PBS group and 1.3 in the HA group. On the other hand, the cross-linked cinnamic acid derivative-introduced HA group had an average of 3.2, and showed a very high water evaporation ratio as compared with the PBS group and the HA group. Furthermore, as apparent from FIG. 16, the water evaporation amount ratio of the crosslinked cinnamic acid derivative-introduced HA group was very stable regardless of the passage of time. This indicates that the crosslinked cinnamic acid derivative-introduced HA stays at the administration site for a long time and exhibits a highly durable effect.
From the above results, GAG having a hydrophobic group introduced via a linking chain, including HA having a cinnamic acid derivative introduced HA and a crosslinked cinnamic acid derivative-introduced HA, is suitable for application to the mucosa and applied to the mucosa. It was shown that it can effectively treat the damage of mucosal epithelial layer. In addition, the effect of the treatment was shown to be highly persistent.
In any of the above animal tests, no adverse effects were observed due to the administration of the cinnamic acid derivative-introduced HA and the crosslinked cinnamic acid derivative-introduced HA. Therefore, the safety of the mucosa application agent of the present invention can be sufficiently estimated. it can.

<Example 15>
(15-1) Preparation of octylamine-introduced sodium hyaluronate 25.8 mg / 2 mL solution of octylamine (ethanol: 0.1 M HCl = 1: 1), 0.1 M of DMT-MM (Wako Pure Chemical Industries, Ltd.) 2 mL of solution (ethanol: water = 1: 1) was added to a solution of sodium hyaluronate (502 mg, 1.25 mmol / disaccharide unit, weight average molecular weight 900,000) in water (50 mL) / ethanol (50 mL). . The mixture was stirred overnight and a 376 mg / 5 mL aqueous solution of sodium bicarbonate (Japanese Pharmacopoeia) was added. After further stirring for 5 hours, the reaction was quenched with acetic acid (107 mg) and sodium chloride (522 mg). Ethanol (250 mL) was added and the resulting precipitate was filtered off and washed twice with 80% ethanol and ethanol in sequence. The solid was dried in vacuo to give a white solid (475 mg). The introduction ratio of octylamine was 12.6% according to HPLC.

(15-2) Preparation of hexadecylamine-introduced sodium hyaluronate
Hexadecylamine 30mg / 3mL solution (ethanol: 0.1M HCl = 1: 1), DMT-MM (Wako Pure Chemical Industries, Ltd.) 0.1M solution (ethanol: water = 1: 1) 1.25mL To a solution of sodium hyaluronate (501 mg, 1.25 mmol / disaccharide unit, weight average molecular weight 900,000) in water (50 mL) / ethanol (50 mL). The mixture was stirred overnight and a 381 mg / 5 mL aqueous solution of sodium bicarbonate (Japanese Pharmacopoeia) was added. After stirring for another 5 hours, the reaction was quenched with acetic acid (107 mg) and sodium chloride (497 mg). Ethanol (250 mL) was added and the resulting precipitate was filtered off and washed twice with 80% ethanol and ethanol in sequence. The solid was dried in vacuo to give a white solid (497 mg). The introduction rate of hexadecylamine was 12% according to HPLC.

(15-3) Preparation of sample solution 64 mg of the compound obtained in (15-1) above was added to 5 mM phosphate buffered saline to make a total volume of 59 ml, and this solution was then shaken overnight on a shaker. . A 0.1 wt% solution of the compound prepared in (15-1) above was obtained.
Similarly, a 0.1 wt% solution of the compound prepared in (15-2) above was obtained.

<Example 16>
Effect of 0.1% cinnamic acid derivative-introduced HA on healing of rabbit corneal epithelium Effect of cinnamic acid derivative-introduced HA prepared in Example 1 on healing of surgically excised rabbit corneal epithelium (surgical model) ).
(16-1) Method 1) Surgical resection of corneal epithelium (surgical model)
After anesthesia by intravenous injection of 5 mg / kg ketamine and 2 mg / kg xylazine and topical administration of 0.4% oxybuprocaine hydrochloride, the central part of the corneal epithelium was placed with trephine (inner diameter 8 mm), 23G needle and It was excised by using a micro scissor.
2) Topical administration 1 hour and 4 hours after peeling the corneal epithelium, 150 μl of physiological saline was administered to the left eye as a control substance, and 0.1% by weight of cinnamon prepared in Example (1-2) above 150 μl of acid derivative-introduced HA solution was administered to the right eye as a test substance. One and two days after exfoliation, the same administration as described above was performed a total of 4 times at 3 hour intervals and 3 days after exfoliation at 3 hour intervals. For administration, a 1 ml injection syringe was used. Fourteen model rabbits with corneal epithelial layer disorder described in 1) above were used as administration subjects.
3) Imaging of corneal epithelial defect Rabbits were given general anesthesia by intravenous injection of 5 mg / kg ketamine and 2 mg / kg xylazine, and then the corneal epithelial disappearance site was treated with 0.2% sodium fluorescein dissolved in PBS. Stained and photographed under UV light. Photographing was performed immediately before administration of the test substance 1 hour after exfoliating the cornea and 3 hours after the second administration 1 to 3 days after exfoliation. At the time of shooting, the focal length was fixed so that the magnification of the photograph was constant.
4) Measurement of corneal epithelial defect The area of the corneal epithelial defect stained with fluorescein sodium was measured on the printed photograph using an image analyzer. It is obtained by subtracting the area of the exfoliation site 3 hours after the final administration 3 days after exfoliation from the area (exfoliation area) of the exfoliation site immediately before administration of the test substance 1 hour after exfoliation of the corneal epithelium. The value was defined as “healing area”.

(16-2) Test results The results of the healing area of each individual are shown in FIG. 17, and the results of the healing rate of each individual are shown in FIG. The healing area and healing rate were calculated as follows.
Healing area = Area after exfoliation-Area at each time point (after 1 to 3 days) Healing rate = Average value of the healing area at each time point (after 1 to 3 days) In FIGS. Compared with the healing area of the corneal epithelium, it was observed that in the eye administered with the 0.1 wt% cinnamic acid derivative-introduced HA solution, it significantly increased at all time points on the 1st to 3rd days. It was also observed that the healing rate was significantly increased in the eyes administered with 0.1 wt% cinnamic acid derivative-introduced HA solution.

<Example 17>
On healing of rabbit corneal epithelium effective surgical resection of 0.1% octylamine introduced HA and hexadecylamine introduced HA on healing of rabbit corneal epithelium, octylamine introduced HA and hexadecylamine prepared in Example 15 Effect of introduced HA (surgical model).
(17-1) Method 1) Surgical resection of corneal epithelium (surgical model)
After anesthesia by intravenous injection of 5 mg / kg ketamine and 2 mg / kg xylazine and topical administration of 0.4% oxybuprocaine hydrochloride, the central part of the corneal epithelium was placed with trephine (inner diameter 8 mm), 23G needle and It was excised by using a micro scissor.
2) Topical administration 1 hour and 4 hours after peeling the corneal epithelium, 150 μl of physiological saline was administered to the left eye as a control substance, and 0.1% by weight of octylamine prepared in the above Example (15-2) 150 μl of introduced HA and hexadecylamine-introduced HA solution were administered to the right eye as a test substance. One and two days after exfoliation, the same administration as described above was performed a total of 4 times at 3 hour intervals and 3 days after exfoliation at 3 hour intervals. For administration, a 1 ml injection syringe was used. Eight model rabbits with corneal epithelial layer disorder described in 1) above were used as administration subjects.
3) Imaging of corneal epithelial defect Rabbits were given general anesthesia by intravenous injection of 5 mg / kg ketamine and 2 mg / kg xylazine, and then the corneal epithelial disappearance site was treated with 0.2% sodium fluorescein dissolved in PBS. Stained and photographed under UV light. Photographing was performed immediately before administration of the test substance 1 hour after exfoliating the cornea and 3 hours after the second administration 1 to 3 days after exfoliation. At the time of shooting, the focal length was fixed so that the magnification of the photograph was constant.
4) Measurement of corneal epithelial defect The area of the corneal epithelial defect stained with fluorescein sodium was measured on the printed photograph using an image analyzer. It is obtained by subtracting the area of the exfoliation site 3 hours after the final administration 3 days after exfoliation from the area (exfoliation area) of the exfoliation site immediately before administration of the test substance 1 hour after exfoliation of the corneal epithelium. The value was defined as “healing area”.

(17-2) Test results The results of the healing area of each individual are shown in FIG. 19, and the results of the healing rate of each individual are shown in FIG. In FIG. 19, C8-L (a: control), C8-R (b), C16-L (c: control), and C16-R (d) were administered with physiological saline as a control for C8-R, respectively. Left eye, right eye administered with 0.1% octylamine-introduced HA solution, left eye administered with saline as a C16-R control, right eye administered with 0.1% hexadecylamine-introduced HA solution To express. In FIG. 20, C8 represents the result of the above test using octylamine, and C16 represents the result of the above test using hexadecylamine. The healing area and healing rate were calculated as follows.
Healing area = Area after exfoliation−Area at each time point (after 1 to 3 days) Healing rate = Average value of the healing area at each time point (after 1 to 3 days) In FIGS. Compared with the healing area of the corneal epithelium, it was observed that in the eyes administered with 0.1 wt% octylamine-introduced HA and hexadecylamine-introduced HA solution, there was a significant increase at all time points on days 1-3. . It was also observed that the healing rate was significantly increased in the eyes administered with 0.1 wt% octylamine-introduced HA or hexadecylamine-introduced HA solution.

FIG. 1 is a diagram showing a spectrum of light transmittance. FIG. 2 is a diagram showing a healing area rate (percentage). FIG. 3 is a diagram showing a healing area. FIG. 4 is a diagram showing the healing rate. FIG. 5 is a diagram showing a healing area. FIG. 6 is a diagram showing the healing rate. FIG. 7 is a diagram showing a healing area. FIG. 8 is a diagram showing the healing rate. FIG. 9 is a diagram showing a healing area. FIG. 10 is a diagram showing the healing rate. FIG. 11a is a diagram showing the persistence of the rabbit corneal epithelium at the site of detachment. FIG. 11 b shows the persistence of the rabbit corneal epithelium at the site of detachment. FIG. 11 c is a diagram showing the persistence of the rabbit corneal epithelium at the site of detachment. FIG. 11d is a diagram showing the persistence of the rabbit corneal epithelium at the site of detachment. FIG. 12a is a diagram showing the persistence of the rabbit corneal epithelium at the site of detachment. FIG. 12 b shows the persistence of the rabbit corneal epithelium at the site of detachment. FIG. 12 c shows the persistence of the rabbit corneal epithelium at the site of detachment. FIG. 12d is a diagram showing the persistence of the rabbit corneal epithelium at the site of detachment. FIG. 13 is a view showing a photograph of the eyeball after ultraviolet irradiation. FIG. 14 is a diagram showing the amount of water evaporated in the isolated cornea. FIG. 15 is a diagram showing the amount of water evaporated in the isolated cornea. FIG. 16 is a diagram showing a change in a water evaporation amount ratio in a model hamster with xerostomia. FIG. 17 is a diagram showing a healing area. FIG. 18 is a diagram showing the healing rate. FIG. 19 is a diagram showing a healing area. FIG. 20 is a diagram showing the healing rate.

Claims (5)

Following formula:
[Wherein R is R ₁ Or R ₂ Represents;
Ac represents an acetyl group;
R ₁ Represents OM or OH, where M represents a metal that forms a pharmaceutically acceptable salt;
R ₂ Is Ph-CH = CH-COO- (CH ₂ ) _1-18 -NH- (wherein Ph represents phenyl)] hyaluronic acid or a salt thereof having a repeating structure of a structural unit represented by the basic skeleton (provided that all R in the hyaluronic acid or a salt thereof) Is R ₁ A corneal epithelial layer disorder preventive or therapeutic agent containing as an active ingredient. The agent for preventing or treating corneal epithelial layer disorder according to claim 1, wherein M is Na. R is R relative to the molar equivalent of the disaccharide repeating unit of hyaluronic acid or a salt thereof. ₂ The corneal epithelial layer disorder preventive or therapeutic agent according to claim 1 or 2, wherein the molar equivalent ratio of the structural unit representing is 5 to 30%. The agent for preventing or treating corneal epithelial layer disorder according to any one of claims 1 to 3, wherein the concentration of the active ingredient is 0.02 to 5% by weight. The agent for preventing or treating corneal epithelial layer disorder according to any one of claims 1 to 4, wherein the concentration of the active ingredient is 0.1 to 0.6% by weight.